'''Rare Metal''' is a tool for gene-based meta-analysis, based upon summary statistics generated from individual data using [ http: //genome.sph.umich.edu/wiki/Rare-Metal-Worker '''Rare Metal Worker'''] .
If you have any questions, please contact: sfengsph at umich dot edu
== Change Log == * Version 0 . 0. 1 released to U of M CSG group. (2/ 13/ 2013) * Version 0. 0. 1 release to public. (2/ 24/ 2013)
== Key Features ==
Rare Metal''' has the following features:* '''Rare Metal''' performs gene-based or region-based meta analysis using Burden tests with the following methods: CMC_counts, Madsen-Browning, SKAT, and Variable Threshold. * '''Rare Metal''' performs single variant metal-analysis by default. * '''Rare Metal''' allows customized groups of variants to be tested.
== Software Download and Installation == * University of Michigan CSG users can go to the following: / net/ fantasia/ home/ sfengsph/ code/ Rare-Metal/ raremetal/ bin/ raremetal
= Where to Download == = * The software package for Linux and Mac (source code included) can be downloaded here: [[Media:raremetal.0.0.2.tgz|software package download]]
=== How to Compile === * Save it to your local path and decompress using the following command: tar xvzf raremetal.0.0.1.tgz * Go to raremetal_0.0. 1/raremetal/src and type the following command to compile: make
=== How to Execute === * Go to raremetal_0.0.1/raremetal/bin and use the following: ./raremetal * For example usage, please refer to [[ http://genome.sph.umich.edu/wiki/Rare-Metal#Example_Usage example command lines]]
== Software Specifications ==
Input Files = == Rare Metal needs the following as input:
==== List of Studies ==== * A file with the path and name of files containing summary statistics generated by raremetalworker should be specified. * If no such file is provided, ''' Rare Metal''' will stop and report FATAL ERROR. * Please go to [[ http: //genome.sph.umich.edu/wiki/Rare-Metal#List_of_Studies_2 example input for study names]] for detailed explanation and examples.
Groups of Variants ==== To perform gene-based or group-based burden test, groups of variants need to be provided. There are two options to provide such information:
=====From Group File =====
* A group file contains the list of groups or genes with the variants to be included in your burden tests.
* Please refer to the instruction of --groupFile option for formats and examples.
From Annotated VCF File ===== * ''' Rare Metal''' allows user to use annotated VCF file as input for grouping of variants, which is optional to input a group file as described above. * '''Rare Metal''' also has the option of generating a VCF file according to the pooled information from individuals studies. Then user can use their favorite annotation tools to annotate the VCF file into the INFO field. Currently, ''' Rare Metal''' only support limited formats of annotated VCF file. * A more flexible way, which is also a recommended way, is to generate a group file from the customized annotated VCF file and use that as input to '''Rare Metal'''. * For formats of annotated VCF that '''Rare Metal''' currently support, please refer to the following [http://genome.sph.umich. edu/wiki/Rare-Metal#Grouping_from_an_Annotated_VCF_File annotated VCF]:
'''NOTE:''' if no grouping method is provided, then only single variant meta analysis will be performed.
=== Software User Interface === The following options are currently available in ''' Rare Metal''':
Options: List of Studies : -- studyName  Grouping Methods : -- groupFile , --annotatedVcf , --annotation , --writeVcf QC Options : --hwe [0. 00], --callRate [0. 00] Association Methods : --burden, --MB, --SKAT, --VT Other Options : --prefix , --maf [0. 05], --longOutput, -- tabulateHits, -- hitsCutoff [1. 0e-06]
==== List of Studies ====* --
studyName option is crucial for ''' Rare Metal''' to work. Ignoring this option will lead to FATAL ERROR and ''' Rare Metal''' will stop.
* The file should contain the path and prefix of the studies you want to include.
* If there is one or more studies that you want to excluded from your list, but want to save some effort of generating a new file, you can put a "#" in front of the line of record. '''
Rare Metal''' will automatically exclude that study from meta analysis. * An example file is in the following:
/net/fantasia/home/sfengsph/prj/raremetal/raremetal/bin/META/TwinsUK/TwinsUK. TG #/net/fantasia/home/sfengsph/prj/raremetal/raremetal/bin/META/HUNT/RareMetalWorker/HUNT_MI_case.TG
* The above example study name file guides '''Rare Metal''' looking for the following files as input (note that the second study has been opt out from the meta analysis, because of the "# " in front of the line)
/net/fantasia/home/sfengsph/prj/raremetal/raremetal/bin/META/TwinsUK /TwinsUK.TG.singlevar.score.txt /net/fantasia/home/sfengsph/prj/raremetal/raremetal/bin/META/TwinsUK/TwinsUK.TG.singlevar. cov. txt
==== Grouping Methods ====
Grouping from a Group File =====
* Grouping methods are only necessary when doing gene-based or group-based burden tests in meta-analysis.
* If none of the grouping method is specified, then only single variant meta-analysis will be performed.
* MARKER_ID must be in the following format:
Here is an example group file:
PLEKHN1 1:901922:G:A 1:901923:C:A 1:902088:G:A 1:902128:C:T 1:902133:C:G 1:902176:C:T 1:905669:C:G
HES4 1:934735:A:C 1:934770:G:A 1:934801:C:T 1:935085:G:A 1:935089:C:G
C1orf159 1:1021285:G:T 1:1021302:T:C 1:1021315:A:C 1:1021386:G:A 1:1022534:C:T 1:1025751:C:T 1:1026913:C:T
Grouping from an Annotated VCF File ===== * If --groupFile option is not specified , ''' Rare Metal''' will look for an annotated vcf file as blue print for variants to group.
* The annotated VCF file should be specified using --annotatedVcf option.
* --annotation should be used with --annotatedVcf together when specific category of functional variants are of interest to be grouped. For example, if grouping nonsynonymous and splicing variants are of interests, the following should be included in command line:
--annotatedVcf your.annotated.vcf --annotation nonsyn/splicing
* Notice that each variant is allowed to have more than one annotations; but each annotation should start with a new key "ANNO=" followed by annotation:genename:other transcript information.
== Generate a VCF File to Annotate Outside of Rare Metal ===== * --writeVCF allows user to write a VCF file including pooled single variants from all studies. Then users can use their favorite annotation tool to annotate the VCF file. After annotating the VCF file, users can use that file as input for '''Rare Metal''' for further gene-based or region-based meta analysis. * The output vcf file will be name as: yourPrefix.pooled.variants.vcf. An example output vcf file is in the following: #CHROM POS ID REF ALT QUAL FILTER INFO 1 115658497 115658497 G A . . ALT_AF=0.380906; 2 74688884 74688884 G A . . ALT_AF=8.33611e-05; 3 121414217 121414217 C A . . ALT_AF=0.0747833; ==== QC Options === = * '''Rare Metal''' allows filtering of variants from individual studies by their HWE pvalue and call rate, which are generated as part of the output from '''Rare Metal Worker'''. * To filter by HWE p-values, --hwe option should be used. The default is 0.0, which means not filtering any of the variants.* To filter by call rate, -- callRate option can be specified. The default is 0.0, which allows no filtering utilized. ==== Association Methods ==== * Currently, four methods are provided in '''Rare Metal''', CMC type burden test, Madsen-Browning burden test, Variable Threshold burden test, and SKAT. ==== Other Options====
* --prefix allows customized prefix for output files.
* --maf specifies the minor allele frequency cutoff when doing gene-based or group-based burden tests. The default is maf<0.05.
* --longOutput allows users to output not only burden test results but also the single variant results (allele frequencies, effect sizes, and p-values) for the variants being grouped together. Please refer to the output files section for detailed explanation and examples.
* --tabulateHits works with --hitsCutoff together to generate reports for genes that have p-value less than specified cutoff from burden tests or SKAT. The default cutoff of p-value for genes to be reported is 1.0e-06, which can be specified by --hitsCutoff option. For more explanations and examples, please go to [[
http://genome.sph.umich.edu/wiki/Rare-Metal# Tabulated_Top_Hits Tabulated Top Hits]].
Output Files ===
==== Single Variant Meta Analysis Output
* Single variant meta analysis output has the following components: header and
* Header lines start with "##" shows summary of the meta analysis including method used, number of studies, and total sample size.
* Header line starts with "#" are column headers for results table.
* An example single variant meta analysis output is shown below:
EFFECT_SIZE: Alternative Allele Effect Size
DIRECTION_BY_STUDY: Effect size direction of alternative allele from each study.
The order of study is consistent with the order of studies listed in the input file for option --
"?" means the variant is not observed or monomorphic from the study.
"!" means the variant observed from this study has different alleles from those
from the first study.
= Burden Tests Meta Analysis Output === = When --longOutput is specified, output includes both burden test results of genes and single variant results of the variants included in burden tests. Otherwise, single variant results of variants included in burden tests will not be included in the output.
= Long Output Format ===== * Here is an example of output file from SKAT when --longOutput is specified.
KLHL17 2 1:897285:A:G;1:898869:C:T 0.0148408,0.00108369 -0.0502034,-0.0256403 0.528269,0.934606 0.00796222 0.00108369 0.0148408 -0.0484494 0.528878
= Short Output Format ===== * Here is an example of output file from SKAT when --longOutput is not specified.
KLHL17 2 1:897285:A:G;1:898869:C:T 0.00796222 0.00108369 0.0148408 -0.0484494 0.528878
Tabulated Top Hits ====
* When --tabulateHits is specified, top hits from Burden tests will be generated. Each method will have an individual tabulated file generated. The purpose of this tabulated file is to list burden test results of top hits together with single variant results from variants being grouped in burden tests. The difference between this file and the standard long-format output file from burden test is that each row of the file represents a single variant that is included in the gene for burden test. This format allows each sorting on users end.
Tabulate top hits will be saved in the file:
yourPrefix.meta.tophits.youMethod.tbl (example files names: TG.meta.tophits.burden.tbl, LDL.meta.tophits.SKAT.tbl)
* According to the example above, PCSK9 had a p-value of 7.54587e-11 from the gene-based burden test, where three variants from this gene were included. Another hit from this meta analysis is APOE, where only one variant was included in the burden test.
Log File ====
* A log file is automatically generated by '''
Rare Metal''' to save the parameters in effect. An example is in the following:
The following parameters are in effect:
--groupFile  --annotatedVcf [
* Here is an example command line to do single variant meta analysis only:
studyName your. studyName. file --prefix yourPrefix
* When you want to do all burden tests using a group file to specify which variants to group:
studyName your. studyName. file --groupFile your.groupfile --burden --MB --SKAT --VT --maf 0.01 --prefix yourPrefix
(NOTE: this will generate single variant meta analysis result and the short format output for burden test results.)
* Here is how to do all SKAT meta analysis using a group file and request a long format output together with tabulated hits:
studyName your. studyName. file --groupFile your.groupfile --SKAT --longOutput --tabulateHits --hitsCutoff 1.0e-07 --prefix yourPrefix
* Here is an example of adding QC filters to variants when doing meta analysis.
studyName your. studyName. file --groupFile your.groupfile --SKAT --longOutput --tabulateHits --hitsCutoff 1.0e-07 --hwe 1e-06 --callRate 0.98 --prefix yourPrefix
* Here is how to do the same thing but reading grouping information from an annotated VCF file:
studyName your. studyName. file --annotatedVcf your.annotated.vcf --annotation nonsyn/stop/splicing --SKAT --longOutput --tabulateHits --hitsCutoff 1.0e-07 --hwe 1e-06 --callRate 0.98 --prefix yourPrefix
* If you want to write a VCF file of pooled variants from all studies, annotate them using your favorite annotation program, and then come back to '''
Rare Metal''' with the annotate VCF file to do burden tests:
First, use the following command to write the VCF file:
studyName your. studyName. file --writeVcf --prefix yourPrefix Second, annotate the VCF file using your favorite annotation program. (Annotated VCF file has to follow the format described here: [[ http://genome.sph.umich.edu/wiki/Rare-Metal# Grouping_from_an_Annotated_VCF_File annotated VCF format]])
Third, use the following command to do meta analysis:
studyName your. studyName. file --annotatedVcf your.annotated.vcf --annotation nonsyn/splicing/stop --burden --MB --SKAT --VT --maf 0.01 --prefix yourPrefix
* For a comprehensive tutorial of
RareMetalWorker and RareMETAL using example data sets, please go to the following:
[http://genome.sph.umich. edu/wiki/Tutorial:_RareMETAL '''RareMETAL Tutorial''']
== Q & A ==